The field of the invention relates to angiotensin converting enzyme 2 (ACE2) and variants of ACE2 for reducing plasma levels of Angiotensin II (1-8) and/or for increasing plasma levels of Angiotensin (1-7) in a subject in need thereof. The disclosed variants of ACE2 may include fragments of ACE2 having ACE2 biological activity for converting AngII (1-8) to Ang (1-7) and having a lower molecular weight than full-length ACE2, which normally is not filtered through the glomerulus and which lower molecular weight permits the fragments of ACE2 to be filtered through the glomerulus. The disclosed variants of ACE2 may be useful for treating conditions that include but are not limited to diabetic and non-diabetic chronic kidney disease, acute renal failure and its prevention, chronic kidney disease, glomerulonephritis, severe hypertension, scleroderma and its skin, pulmonary, kidney and hypertensive complications, malignant hypertension, renovascular hypertension secondary to renal artery stenosis, idiopathic pulmonary fibrosis, liver fibrosis such as in liver cirrhosis patients, an aortic aneurysm, cardiac fibrosis and remodeling, left ventricular hypertrophy, and an acute stroke.
Activation of the renin angiotensin system (RAS) plays a major role in the pathogenesis of hypertension, cardiovascular disease, diabetic kidney disease and the progression of CKD to ESRD1-3. Moreover, in acute renal failure the RAS is also activated4-7. There is a need for new approaches to counteract RAS over-activity that expand and improve on the existing approaches based primarily on blocking formation of Ang II formation or blocking the action of Ang II. We have been at the forefront of proposing therapies aimed at promoting the degradation of Ang II8-13. An important biological effect of ACE2 is to convert AngII(1-8) to Ang(1-7), a process that tends to lower AngII(1-8) and therefore prevents the potentially detrimental actions of this peptide. In addition, Ang(1-7) is formed as a result of Ang II(1-8) cleavage and this peptide, by directly activating the Mas receptor, has tissue protective functions that are generally opposite to those of AngII(1-8). Indeed, there is increasing evidence that Ang(1-7) has a vast array of potential therapeutic applications and this also emphasizes the importance of Ang(1-7) forming enzymes as potential therapeutic targets with the dual advantage of degrading Ang II and forming Ang(1-7).
Years ago we and others have purified and produced murine ACE2 as a way to circumvent the immunogenicity14 that we observed in our initial studies using for the first time human ACE2 given to mice with hypertension induced by AngII infusions13. In recent studies we examined the kidney effects of murine recombinant ACE2 given to mice with streptozotocin-induced diabetic kidney disease. (See Wysocki et al., Angiotensin-converting enzyme 2 amplification limited to circulation does not protect mice from development of diabetic nephropathy,” Kidney Int. 2016 Dec. 4. Pii: S0085-2538(16)30565-8, the content of which is incorporated herein by reference in its entirety). Two approaches were used in this study: amplification of circulating ACE2 by intraperitoneal daily injections for 4 weeks and by ACE2 gene delivery15. Delivery of ACE2 using minicircles resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute Ang II(1-8) load. In mice with STZ-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. Albuminuria, glomerular mesangial expansion, glomerular cellularity and glomerular size, were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls10. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic kidney disease despite months of sustained very high plasma ACE2 levels. These findings emphasize the importance of targeting the kidney rather than the circulatory renin angiotensin system to combat early stages of diabetic kidney disease and kidney disease in general. The large molecular size of recombinant ACE2 renders it non-filterable by a normal glomerulus or in early forms of kidney disease, a time critical to intervene to prevent disease progression In more advanced glomerular kidney disease, by contrast, we have been able to show that infused rACE2 can be recovered in the urine10. At this late stage of advanced disease, it is difficult to reverse kidney alterations and reverse fibrosis. Therefore, to circumvent this limitation we designed shorter forms of ACE2 that are much more suitable to treat kidney disease and provide better tissue penetration to other organs such as lungs and the heart.
Based on our findings we have created forms of ACE2 of shorter molecular size that are deliverable to the kidney prior to the development of marked alterations in glomerular permeability and better delivered to the kidney in all instances. ACE2 is typically observed as a 110 kD protein which is not filterable by the kidney and appears in the urine as a shedding product from the renal apical tubular membrane of the kidney where ACE2 is abundantly expressed9-11,16. We have developed smaller molecular weight recombinant ACE2 proteins that are very active. This means that they retain full activity and potential therapeutic use when the goal is to increase ACE2 activity not only in the systemic circulation, just like it is done by the already available human recombinant intact ACE2, but also rather they are unique in that their smaller size makes them deliverable to the kidney by glomerular filtration and thus better for the treatment of kidney disease and tissue penetration of other organs as well.
We have shown that decreasing the size of ACE2 renders it easily filterable through the glomerular barrier in states of mild increases in glomerular permeability, such as acute kidney injury or in early phases of diabetic kidney disease i.e. microalbuminuric stage. The overarching goal is to develop a form of shorter ACE2 that can be delivered easily to the kidney and therefore combat kidney disease This approach is distinctive and complimentary to currently used ACE inhibitors and AT1 blockers. We postulate that enhancing the degradation of Ang II offers the distinctive advantage of leading to the formation of Ang 1-7, a renoprotective peptide, and is also a more natural physiologic approach than blocking the formation or action of Ang II or its receptors as currently done with existing agents. As a way to increase tubular reabsorption of the short ACE2 fragments filterable through the glomerulus and therefore enhance their kidney uptake, the short ACE2 fragments will be conjugated to low molecular fusion polypeptides. These fusion polypeptides include, but are not limited to, Fc (constant fragment of human IgG), the DIII domain of human serum albumin and lysozyme. All of those polypeptides have been shown to be reabsorbed on apical surface of the kidney tubules by receptor-mediated endocytosis. The subject matter of this application is discussed further herein.